Determination of allele frequencies by dna pooling and ld between snPs

T

Figure 5 Position of SNPs at the COMT locus and differences in allele frequencies seen between schizophrenic patients and control individuals generated from DNA pools (Shifman 2002)

welve SNPs in COMT gene were tested to understand if any of these were associated with schizophrenia. Among different SNPs chosen from public databases, there was extensively studied Val/Met polymorphism at rs165688. Firstly, 12 different SNPs were examined by accurate measurement of allele frequencies of individuals from DNA pools. Allele frequencies were compared between schizophrenic patients and controls by Pyrosequencing technique. Four SNPs of the twelve have not shown a polymorphism in the defined homogenous population. One SNP was also excluded from study due to inability to undergo Polymerase chain reaction amplification. There was a significant difference in allele frequencies between controls and patients for remaining SNPs (Figure 5). Genotypes of seventy individuals were randomly picked out and examined for each of these SNPs. It was found that SNPs rs6269 and rs4633 were associated in high linkage disequilibrium with other genetic variant of Val/Met polymorphism rs165688 (also known as rs4680) (Shifman 2002).

Determination of genetic association with schizophrenia by employing individual genotyping

Well studied SNP rs165688 encoding Val/Met polymorphism was genotyped in 720 schizophrenic patients and 2970 control individuals (Table 1). What results showed was a significant association between SNP rs165688 and schizophrenia among male individuals homozygous for Val allele, which have shown an excess of G/G genotype levels. Heterozygous individuals (A/G) and homozygous for Met allele (A/A) haven’t shown a significant alteration in the frequencies of COMT genotype among patients and healthy individuals.

Another SNP rs165599 was analysed in 724 patients with schizophrenia and 4014 control individuals. The results showed significant differences found in allele frequencies between men and women in the control samples. Schizophrenic female patients homozygous for high activity Val allele showed an increase in G/G genotype levels in comparison to healthy individuals. Men have shown smaller effect of SNP rs165599 in the association with schizophrenia.

SNP rs737865 was genotyped in 714 patients with schizophrenia and 2849 control individuals. What results showed was a significant association of rs737865 with schizophrenia in men and women. Given the observed genotype frequencies, male patients homozygous for high activity allele showed significant increase in G/G genotype levels, female schizophrenic individuals showed an increase in A/G and G/G genotypes. Although the allele frequencies in male and female were similar, genotype distribution showed different results between the sexes (Shifman 2002).

			Frequency (%)
SNP And Sex	Sample size		G		G/G		A/G		A/A		P*a
	Control	Patient	Control	Patient	Control	Patient	Control	Patient	Control	Patient	Genotype	Allele	G/G
rs165688:
F	706	262	50.8	53.4	25.9	26.3	49.7	54.2	24.4	19.5	.25	.30	.90
M	2,264	458	52.1	55.8	25.8	31.9	52.6	47.8	21.6	20.3	.027	.041	.0074
All											.041	.024	.023
rs165599:
F	1,368	263	34.7	44.9	11.7	45.9	45.6	45.6	42.3	32.3	.000010	.0000091	.0000068
M	3,519	461	39.0	41.8	14.8	17.8	48.4	47.9	36.8	34.3	.20	.10	.090
All											.000030	.000088	.000072
rs737865:
F	685	250	38.7	45.0	15.5	17.6	46.4	54.8	38.1	27.6	.012	.014	.04*b
M	2,164	464	40.9	45.6	15.8	22.8	50.2	45.5	34.0	31.7	.0011	.0083	.00023
All											.00016	.00036	.00048
a – P values are provided for testing genotype, allele, and G/G versus A/G + A/A frequencies in the patients against those in the control individuals. b – In woman, a stronger P value was obtained when testing A/G and G/G versus A/A (P=.029).

Table 1 Genotyping results of an individual and determination of genetic association with schizophrenia (Shifman 2002).